Glycine confers neuroprotection through microRNA-301a/PTEN signaling

Glycine is known to protect against neuronal death. However, the underlying mechanism remains to be elucidated. The microRNA-301a is involved in both biological and pathological processes. But it is not known whether microRNA-301a has a neuroprotective property. In this study, we aimed to determine whether glycine-induced neuroprotection requires microRNA-301a-dependent signaling. We provided the first evidence that glycine increased the expression of microRNA-301a in cultured rat cortical neurons and protected against cortical neuronal death through up-regulation of microRNA-301a after oxygen-glucose deprivation. MicroRNA-301a directly bound the predicted 3′UTR target sites of PTEN and reduced PTEN expression in cortical neurons. We revealed that PTEN down-regulation by microRNA-301a mediated glycine-induced neuroprotective effect following oxygen-glucose deprivation. Our results suggest that 1) microRNA-301a is neuroprotective in oxygen-glucose deprivation-induced neuronal injury; 2) glycine is an upstream regulator of microRNA-301a; 3) glycine confers neuroprotection through microRNA-301a/PTEN signal pathway.


Background
Glycine is the simplest non-essential amino acid, which is a critical building block in many proteins. Glycine is essential for the synthesis of many biomolecules such as creatine, porphyrins and purine nucleotides. Glycine plays a fundamental role in cell metabolism [1,2]. In the adult CNS, glycine is a major inhibitory neurotransmitter that binds to glycine receptor (a chloride-permeable ion channel) to inhibit postsynaptic neurons [3][4][5][6][7]. Interestingly, glycine is also a co-agonist of the excitatory NMDA receptor, a calcium-permeable ion channel in the CNS [8,9]. Glycine has been shown to have neuroprotective effect in a variety of experimental models including ischemiareperfusion injury, anoxia, hypoxia, ROS, chemically induced energy depletion [10][11][12][13]. In double-blinded, placebo-controlled clinical trial, glycine treatment shows significantly improved outcome and tended to decrease the 30 day mortality of stroke patients [14]. However, how glycine exerts its neuroprotective effect is largely unknown.
MicroRNAs (miRNAs) are a class of non-coding RNAs (∼22 nucleotides) that negatively regulate protein expression [37,38]. MiRNAs regulate the expression of at least one third of the human genome and play a critical role in a variety of normal biological processes including cell differentiation, apoptosis, cell development and cell metabolism [37,39,40]. In animals, miRNAs regulate mRNA translation via imperfect pairing with nucleotide sequences within the 3′-untranslated region (3′UTR) of target genes [41].
MiRNA-301a (miR-301a) is the member of miR-130/ 301a family. miR-301a has been shown to involve in some biological and pathological processes, including cell development, cell differentiation, inflammation, apoptosis and cancer [42][43][44][45]. miR-301a is up-regulated in pancreatic cancer and to activate NF-kB by negative regulation of NF-kB-repressing factor gene [46]. miR-301a contributes to IL-6-induced insulin resistance by direct regulation of PTEN and downstream Akt signaling [47]. Recent studies demonstrate that miR-301a promotes cancer cell metastasis in breast, hepatocellular and gastric tumors through different target genes [43,44,48]. However, it is not clear whether miR-301a plays a role in neuronal survival.
In the present study, we demonstrate that glycineinduced neuroprotection depends on miR-301a and its downstream signaling. We show that the expression of miR-301a is elevated by glycine. miR-301a mediates glycine-induced neuroprotective effect in cultured cortical neurons subjected to oxygen-glucose deprivation (OGD). miR-301a binds the predicted 3′UTR target sites of PTEN and reduces PTEN expression. We provide evidence that glycine-induced neuroprotection is mediated through the up-regulation of miR-301a and subsequent suppression of PTEN expression.

Glycine is neuroprotective in OGD-induced cortical neuronal injury
Glycine has been shown to protect against OGD-induced neuronal injury. To verify the neuroprotective role of glycine in our experimental conditions, we examined the effect of glycine on cultured cortical neurons subjected to OGD. The cultured cortical neurons were injured by OGD for 1 h. At 1 h after OGD insult the cortical neurons were treated with glycine (100 μM). We treated the neurons with glycine at 1 h after OGD, instead of before OGD, in this study because of its clinical relevance. It is generally accepted that ischemic stroke treatment is required to be done within 4.5 h after ischemia/ reperfusion onset. The treated neurons were collected for cell death and viability assays at 24 h after OGD insult. As shown in Fig. 1, LDH, MTT and FDA labeling assays showed that treatment of glycine reduced OGDinduced cortical neuronal death. These results support Fig. 1 Glycine protects against OGD-induced cortical neuronal death. a, Glycine (10-300 μM) reduces OGD-induced increase of LDH release (n = 6, *P < 0.05 vs. Sham; # P < 0.05 vs. Inj, ANOVA test). b, MTT assay shows that glycine (10-300 μM) increases neuronal survival rate in OGD-induced cortical neuronal injury (n = 6, *P < 0.05 vs. Sham; # P < 0.05 vs. Inj, ANOVA test). c, Representative images of FDA labelling in cortical neuronal cultures showing that glycine (100 μM) reduces OGD induced-neuronal death. Inj, injury; Gly, glycine a neuroprotective role of glycine in OGD-induced neuronal injury.

Glycine increases miR-301a expression in cortical neurons
To determine whether glycine exerts its neuroprotective effect through regulating miRNA-dependent signaling, we performed microarray to analyze the miRNA expression profiles in cultured rat cortical neurons following glycine treatment. The cultured neurons were treated with or without glycine (100 μM) for 24 h and then collected for miRNA microarray assays. Total RNAs were isolated from glycine-treated cortical neurons and the miRNA microarray profiling of rat miRNA genes was performed (Fig. 2a). We found that glycine treatment resulted in differential changes in the expression of miRNAs in the cortical neurons (Fig. 2a), among which the miR-301a was markedly increased (Fig. 2b).
To provide further evidence to verify glycine-induced increase of miR-301a, qRT-PCR was used to measure the miR-301a expression after the rat cortical neurons were treated with glycine (100 μM) for 24 h. As expected, the qRT-PCR results confirmed that glycine enhanced the expression of miR-301a in the cortical neurons (Fig. 3).

miR-301a is neuroprotective in OGD-induced neuronal injury
Given the neuroprotective effect of glycine, the observed up-regulation of miR-301a by glycine led us to reason that miR-301a may play a neuroprotective role in neuronal injury. We first set up to measure the response of endogenous miR-301a to neuronal injury. We examined the expression of miR-301a in cultured cortical neurons at different time after OGD insult. The qRT-PCR data revealed that the expression of miR-301a, but not miR-146a, was decreased at 2, 6, 12 and 24 h after OGD-induced injury in cultured cortical neurons (Fig. 4a, b).
To determine whether miR-301a is neuroprotective, the cortical neurons were treated with miR-301a agomir or agomir control. At 24 h after the treatment, the neurons were subjected to OGD for 1 h. At 24 h after OGD insult, we found that treatment of miR-301a agomir, but not the agomir control, protected against OGD-induced neuronal death after OGD insult (Fig. 4c). In the same experimental protocols, treatment of miR-301a antagomir but not antagomir control increased OGD-induced neuronal death (Fig. 4d). These results provides the first evidence that miR-301a is neuroprotective in neuronal injury.

miR-301a is required for glycine-induced neuroprotection
To determine whether glycine-induced neuroprotection is mediated through miR-301a, we first measured the effects of glycine on the expression of miR-301a in cultured cortical neurons at different time following OGD insult. The neurons were subjected to OGD for 1 h and treated with glycine (100 μM) at 1 h after OGD. The qRT-PCR data showed that glycine prevented OGDinduced decrease of miR-301a at 2, 6, 12 and 24 h after injury in cultured cortical neurons (Fig. 5a). To provide evidence whether glycine-induced increase of miR-301a conferred neuroprotection, the cortical neurons were treated with miR-301a antagomir or antagomir control. At 24 h after treatment, the treated neurons were subjected to OGD for 1 h. At 1 h after OGD injury the cortical neurons were treated with glycine (100 μM) and collected for cell death and viability assays at 24 h after OGD insult. We showed that suppression of miR-301a by miR-301a antagomir attenuated glycine-induced neuroprotective effect (Fig. 5b). To provide further evidence, we treated the neurons with miR-301a agomir or agomir control. At 24 h after the treatment, the neurons were subjected to OGD for 1 h and then treated with glycine (100 μM). At 24 h after OGD insult, we showed that the neuroprotective efficiency induced by glycine in agomir-treated neurons was not significantly higher than that in agomir controltreated neurons (Fig. 5c), suggesting that miR-301a is in the same pathway with glycine to exert neuroprotective effect. Collectively, these data indicate that glycine confers neuroprotection through enhancing miR-301a expression.

PTEN is a target gene of miR-301a
To explore how miR-301a exerts its effect in mediating glycine-induced neuroprotection, we analyzed the target genes of miR-301a by miRanda (www.microrna.org), TargetScan (www.targetscan.org) and miRDB (mirdb.org) and predicted PTEN as a target of miR-301a. As shown in Fig. 6a, miR-301a was predicted to target the 2206-2212 nts of PTEN 3′UTR. To determine whether miR-301a directly binds the predicted 3′UTR sites of PTEN, we made a reporter construct harbouring the 589 bp fragment of PTEN 3′UTR flanking the entire putative target sequence. We performed luciferase assay and showed that ectopic expression of miR-301a agomir resulted in a significant reduction of luciferase activity in the PC12 cells (Fig. 6b). The mutation of the seed sequence of miR-301a within the 3′UTR of PTEN abrogated the inhibition of luciferase activity by exogenous miR-301a agomir in PC12 cells (Fig. 6b). In contrast, the luciferase activity was increased in the PC12 cells co-transfected with a luciferase reporter containing the PTEN-3′UTR (pMIR-PTEN 3′UTR) and miR-301a antagomir (Fig. 6c). These results suggest that miR-301a directly binds the predicted 3′UTR target sites of PTEN.

PTEN is regulated by miR-301a in cortical neurons
To determine the functional consequence of miR-301a binding to PTEN, we tested the effects of miR-301a agomir or antagomir on the mRNA and protein expression The cortical neurons isolated from 3 rats were used for the assay. b, The data of microarray assay show that the expression of miR-301a is remarkably increased in glycine (100 μM)-treated cortical neurons (n = 3, *P < 0.05 vs. Control, ANOVA test). The levels of miR-301a detected by microarray were normalized to a negative control of U6 snRNA of PTEN in cortical neurons. We demonstrated that the level of PTEN mRNA was down-regulated in the cultured cortical neurons treated with miR-301a agomir for 24 h and up-regulated in the cultured cortical neurons treated with miR-301a antagomir for 24 h (Fig.7a). Western blot analysis further showed that 24 h treatment of miR-301 agomir decreased the protein expression of PTEN but 24 h treatment of miR-301 antagomir increased the level of PTEN proteins in cultured cortical neurons (Fig. 7b-c). These data indicate that miR-301 negatively regulates the mRNA and protein expression of PTEN in cortical neurons.

miR-301a suppresses PTEN to mediate glycine-induced neuroprotection
We next tested whether miR-301a mediates glycine-induced neuroprotection through PTEN down-regulation. The cultured cortical neurons were transduced with lentiviral vectors encoding PTEN cDNAs. At 48 h after transduction, the neurons were subjected to OGD for 1 h. At 1 h after OGD injury the cortical neurons were treated with glycine (100 μM) and collected for cell death and viability assays at 24 h after OGD insult. We showed that overexpression of PTEN reduced glycine-induced neuroprotection (Fig. 8a).
The cortical neurons were also transduced with lentiviral PTEN siRNAs. At 48 h after transduction, the neurons were subjected to OGD for 1 h. At 1 h after OGD insult the neurons were treated with glycine (100 μM) and collected for cell death and viability assays at 24 h following OGD insult. We found that pre-suppression of PTEN by the siRNA did not significantly enhance glycineinduced neuroprotective effect (Fig. 8b).
The cortical neurons were then treated with lentiviral PTEN siRNA or miR-301a antagomir. The neurons were first treated with lentiviral PTEN siRNA. At 24 h after the transduction, the neurons were treated with miR-301a antagomir or antagomir control. At 24 h after the treatment of antagomir or antagomir control, the neurons were subjected to OGD for 1 h. At 1 h after OGD insult the neurons were treated with glycine (100 μM) and collected for cell death and viability assays at 24 h after OGD insult. We demonstrated that PTEN down-regulation by PTEN siRNA prevented miR-301a antagomir from blocking glycine-induced neuroprotection (Fig. 8c). Taken together, we conclude that miR-301a targets PTEN to mediate glycine-induced neuroprotection.

Discussion
Glycine is shown to confer protection against neuronal injuries in both in vitro and in vivo experimental conditions [10-13, 49, 50]. Importantly, clinical trial has shown that glycine treatment improves outcome of ischemic stroke patients [14]. However, how glycine exerts its neuroprotective effect remains to be elucidated. The present study provides new evidence that the neuroprotective effect of glycine is mediated by the enhancement of miR-301a expression and subsequent suppression of PTEN expression in cortical neurons. These findings reveal a molecular mechanism by which miRNA-dependent signaling mediates glycine-induced neuroprotection. It is not yet clear how miR301a is regulated by glycine. The expression level of biologically active mature miRNAs is the result of a fine mechanism of biogenesis, carried out by different enzymatic complexes that exert their function at transcriptional and post-transcriptional levels [51]. It is possible that glycine may regulate the processing of miRNA biogenesis. In the cytoplasm, Dicer is an RNase III that digests the pre-miRNA into a 20-25 nucleotides mature duplex miRNA [51]. For example, TAp63, a p53 family member, has been reported to coordinately regulate Dicer and miR-130b to suppress metastasis [52]. TAp63 binds to and transactivates the Dicer promoter, resulting in a direct transcriptional regulation of Dicer by TAp63 [52]. We would reason that glycine may regulate miR301a through a transcriptional mechanism by which Dicer is modulated. Further studies will be performed to test this hypothesis.
As glycine is a co-agonist of NMDA receptors, regulation of miR301a by glycine may be mediated by NMDA receptor-dependent signaling. Previous study has provided evidence that the activation of GluN2A-containing NMDA receptors, but not GluN2B-containing NMDA receptors, contributes to glycine-induced neuroprotective effect in ischemic injury [10,50]. These studies show that glycine induces its neuroprotection through enhancement of Akt activation and CREB phosphorylation. Thus, it is necessary to investigate next whether GluN2A-containing NMDA receptor-dependent Akt/forkhead/FOXO and/or Fig. 3 Verification of glycine-induced increase of miR-301a by qRT-PCR. qRT-PCR reveals that treatment of glycine (100 μM) for 24 h increased the expression of miR-301a in cultured cortical neurons (n = 6, *P < 0.05 vs. Control, ANOVA test). The levels of miRNAs were calculated using U6 snRNA as an internal control CREB signaling mediate glycine-induced modulation of miR-301a.
Recent studies indicate that miR-301a plays a critical role in tumorigenesis [43,44,46,48]. It has been reported that miR-301a is up-regulated in hepatocellular carcinoma and modulates NF-kB expression by negatively regulating Gax [53]. miR-301a is also up-regulated in gastric tumor cells and involved in the clinical progression and prognosis of gastric cancer [45]. miR-301a promotes migration and invasion by targeting TGFBR2 in human colorectal cancer [48]. The up-regulation of miR-301a in breast cancer and Ewing's sarcoma cells promotes tumor metastasis and tumor cell proliferation by targeting the tumor suppressor PTEN [44,54]. Through negative regulation of SMAD4, miR-301a promotes pancreatic cancer progression [55]. In addition to its effect on cancer, miR-301a is shown to play an important role in regulating the expression of Kv4.2 in diabetes [56] and controlling autoimmune demyelination by regulating the T-helper 17 immune response [57]. Our study for the first time demonstrates that miR-301a exerts a neuroprotective effect in OGD-induced neuronal injury, implicating a therapeutic potential of miR-301a signaling in neurological diseases. Interestingly, we reveal that glycine acts as an upstream regulator to enhance miR-301a expression, which confer neuroprotection in cortical neurons.

MTT Assay
The viability of the cells in the neuronal cultures was assessed by their ability to uptake thiazolyl blue tetrazolium bromide (MTT). The cells were incubated with MTT for 1 h, then lysed with dimethyl sulfoxide (DMSO) and left at room temperature in the dark overnight. The lysates were then read on a plate reader (PowerWave X, Bio-Tek) at the absorbance wavelength of 540 nm.

Real-time qRT-PCR
For the quantification of miRNAs or mRNA, total RNAs were isolated using TRIzol reagent (Invitrogen, USA), the reverse transcribed using Maxima H Minus First Strand cDNA Synthesis Kit (Thermo, USA) and the qPCR reaction using SYBR Green qPCR Master mixture (ABI, USA). The reverse transcription was performed on Bio-Rad MJ Mini Instrument (Bio-Rad, USA) and qPCR was performed on QuantStudio 6 K Flex Instrument (ABI, USA).

Constructs and treatment
The miR-301a agomir, agomir control, antagomir, antagomir control were purchased from RioBio (China). The cultures were washed with ECS for 10 min, and then treated with serum-free medium supplemented with 1.0 μM agomir, agomir control, antagomir or antagomir control for 2 h at 5 % CO2, 95 % humidity and 37°C. After incubation, the cultures were further cultured in maintenance medium for 24 h [60].
The lentiviral PTEN cDNA particles and lentiviral PTEN siRNA particles were purchased from GeneChem (China). The treatment of particles was performed in cultured cortical neurons based on the manufacturer's instructions.
A total of 5 × 10 4 PC12 cells were seeded in 24-well plates for 24 h and then co-transfected with 100 ng pMIR-PTEN-3′UTR constructs using Lipofectamine 2000 (Invitrogen, USA) with 1.0 μM miR-301a agomir or agomir control, or with the miR-301a antagomir or antagomir control. Renilla luciferase pRL-TK reporter (Promega, USA) was co-transfected to monitor the transfection efficiency. At 24 h post-transfection, the luciferase activities were measured by the dual-luciferase reporter assay system using the Glomax2020 luminometer (Promega, USA). Data were normalized by Renilla luciferase. Each experiment was performed at least three times in triplicate wells.

Western blotting
Western blotting assay was performed as described previously [18,58]. For the detection of phospho-Akt, the samples prepared in the same day were used. The polyvinylidene difluoride membrane (Millipore, USA) was incubated with primary antibody against PTEN and βactin (Cell Signaling, USA). Primary antibodies were labeled with horseradish peroxidase-conjugated secondary antibody, and protein bands were imaged using Super-Signal West Femto Maximum Sensitivity Substrate (Pierce, USA). The EC3 Imaging System (UVP, USA) was used to obtained blot images directly from the polyvinylidene difluoride membrane. The quantification of Western blot data was performed using ImageJ software.

Statistical analysis
Student's t test or ANOVA test was used where appropriate to examine the statistical significance of the differences between groups of data. Newman-Keuls tests were used for post-hoc comparisons when appropriate.
All results are presented as mean ± SE. Significance was placed at P < 0.05.

Conclusions
Our results indicate that microRNA-301a is neuroprotective in oxygen-glucose deprivation-induced neuronal injury. We provide evidence that glycine is an upstream regulator of microRNA-301a. We conclude that glycine confers neuroprotection through microRNA-301a/PTEN signal pathway.